The activity and mode of action of the new nucleoside analog (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) against varicella-zoster virus (VZV) were determined. In cell culture, replication of different strains of VZV was inhibited to 50% by 0.4 to 0.7 ,uM 2HM-HBG, while 685 ,uM was required to inhibit 50% of the DNA synthesis in uninfected human lung fibroblasts. A thymidine kinase-negative VZV strain was not inhibited by 100 pM 2HM-HBG. Inhibition of'VZV replication was not reversible after 7 to 14 days of incubation, depending on the multiplicity of VZV. 2HM-HBG was shown to be selectively phosphorylated by purified VZV thymidine kinase, with an inhibition constant of 32.5 ,uM. The antiviral activity of 2HM-HBG in cell culture was decreased by the addition of deoxythymidine and deoxycytidine but not by other ribo-or deoxvribonucleosides.The new acyclic guanosine analog (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) exhibits structural similarities to 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV] [3]). It was therefore considered that the two compounds may have similar mechanisms of action. These include selective phosphorylation by herpesvirusinduced thymidine kinases (TK) to monophosphates and, after cellular phosphorylation to triphosphates, inhibition of viral DNA synthesis. PPi analogs, on the other hand, inhibit viral DNA polymerase activity directly (9). They are therefore also active against herpesviruses that do not encode a specific TK (19). The most active PPi analog is Foscamet.The aim of our study was to describe the antiviral activity of 2HM-HBG in cell culture against varicella-zoster virus (VZV), its cellular toxicity, phosphorylation by VZV TK, reversibility of the antiviral activity by exogenous addition of natural nucleosides, and the probable mechanism of action. MATERIALS AND METHODSCells. Human embryonic lung (HL) fibroblasts were cultured in Eagle minimal essential medium with 2% calf serum and antibiotics. These cells were chosen for their low deoxythymidine (dThd) content, which is known to influence the sensitivity to certain antiviral substances (5).Virus. VZV strains ULF and 9/84 were obtained from vesicles of patients with clinical varicella infections and isolated at the National Bacteriological Laboratory, Stockholm, Sweden. The VZV strain pE, an isolate from a patient with clinical varicella, was obtained from the

The separate (+) and (−) enantiomers of the acyclic guanosine analogue 9-[4-hydroxy-2-(hydroxymethyl)-butyl]guanine (2HM-HBG) were tested for inhibition of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). In all cases the (−) enantiomer was the most active enantiomer. The (+) enantiomer was 10 times less active than the racemate against VZV and inactive against HSV-1 and -2. The parent compound, 9-(4-hydroxybutyl)guanine, containing an unbranched side chain, was inactive against VZV, whereas substitution with a hydroxymethyl group at the 2 or 3 position of the side chain conferred anti-VZV activity. The effect of hydroxymethyl substitution may increase the recognition of the compound by the VZV thymidine kinase by increasing its similarity to the natural substrate thymidine. Further substitution of the side chain of the parent compound with oxygen, fluorine or hydroxyl groups did not confer antiviral activity against VZV. Two VZV strains were isolated which could be grown in the presence of high concentrations of 2HM-HBG and which were also cross-resistant to other nucleoside analogues. These strains induced very little viral thymidine kinase activity in infected cells and thus were probably deficient for a functional thymidine kinase. 2HM-HBG exhibited a persistent antiviral effect even after the nucleoside was removed from the medium of VZV-infected cells, whereas acyclovir did not show this effect.

The acyclic guanosine analog (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, (+-)2HM-HBG, is an effective inhibitor of herpes simplex virus and varicella-zoster virus infections in vitro. This report is concerned with the pharmacokinetic evaluation of the drug in rats and monkeys and its antiviral activity in African green monkeys infected with simian varicella virus (SVV), a virus closely related to varicella-zoster virus that is also susceptible to inhibition by (+)2HM-HBG. Elimination half-lives in plasma following intravenous administration to monkeys (100 ,mol/kg of body weight) ranged from 1.8 to 2.2 h, and total body clearance was 9.0 ± 0.4 ml/min per kg (mean ± standard error). After oral administration, levels in plasma were low, with a maximum concentration of the drug of only 3.1 ± 0.8 ,M, a time to reach maximum concentration of drug of 2.7 ± 0.4 h, and an oral bioavailability of 10.6 ± 1.4%. Because of the low oral bioavailability, SW-infected monkeys were treated intramuscularly with (±)2HM-HBG. (±)2HM-HBG at a dosage of 10 mg/kg of body weight per day allowed moderate viremia, whereas a dosage of 30 mg/kg of body weight per day strongly suppressed viremia with minimal numbers of virus plaques fromn blood specimens collected at days 3, 5, and 7 postinfection and complete clearance at day 9 postinfection. Titers of antibody to SVV were also low. Treatment three times daily was somewhat more efficacious than treatment twice daily. Thus, (±)2HM-HBG is an effective inhibitor of SW replication in vivo, despite the fact that levels of (-)2HM-HBG in plasma were low at extended periods of time and below the concentration of drug giving 50% inhibition of plaque formation obtained in vitro.

The triphosphates of the antiherpesvirus acyclic guanosine analogs 9-[4-hydroxy-2-(hydroxymethyl)butyl] guanine (2HM-HBG), 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]), and 9-(3,4-dihydroxybutyl)guanine (buciclovir) were examined for their effects on partially purified varicella-zoster virus (VZV) DNA polymerase as well as cellular DNA polymerase a. The triphosphate of 2HM-HBG competitively inhibited the incorporation of dGMP into DNA catalyzed by the VZV DNA polymerase. 2HM-HBG-triphosphate (2HM-HBG-TP) had a higher affinity for the dGTP-binding site on the VZV DNA polymerase than did dGTP; apparent Km and Ki values of dGTP and 2HM-HBG-TP were 0.64 and 0.034 p,M, respectively. ACVtriphosphate (ACV-TP) was found to be the most potent inhibitor of VZV DNA polymerase. ACV-TP had a 14 and 464 times better direct inhibitory effect than 2HM-HBG-TP and buciclovir-triphosphate, respectively. The cellular (human embryonic lung fibroblast) DNA polymerase a inhibition was related to viral polymerase inhibition as efficacy ratios: 2HM-HBG-TP had a ratio of more than 1,000, which appeared to be similar to that of ACV-TP.

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